Hemolysis and the intravascular release of hemoglobin S are central to the pathophysiology of sickle cell disease (SCD). During the current funding period, we showed that heme derived from sickle red blood cells acts as a damage-associated molecular pattern that can activate toll-like receptor 4 (TLR4) of the innate immune system, independently of its cognate ligand lipopolysaccharide, leading to oxidant production, rapid P-selectin and von Willebrand factor expression on endothelium, and vaso-occlusion (VO) in SCD mice. We hypothesize that the innate immune system, including TLR4 and complement, is fundamental to understanding hemolysis-driven inflammation, coagulation, VO, and the cumulative organ pathology in SCD. The first aim of our proposal is to identify the heme- binding site on the myeloid differentiation factor 2 (MD-2)/TLR4 complex. To date, no studies have examined the heme-binding site on the MD-2/TLR4 complex. Identifying the heme-binding site on MD-2/TLR4 is vital for developing therapies to interrupt heme-driven inflammation and VO. The second aim will examine the impact of global Tlr4 deficiency and the specific contribution of TLR4 in leukocytes, platelets, and the vessel wall to SCD pathogenesis. Recent studies by our group and others used Tlr4-/-, non-sickle cell mice transplanted with Tlr4+/+ sickle bone marrow to underscore the importance of TLR4 in the vessel wall in promoting VO and acute chest syndrome in SCD. However, the impact of Tlr4 deficiency on inflammation, coagulation, and cumulative organ pathology has not been tested in a global Tlr4-/- SCD mouse model. We will test the hypothesis that TLR4 is a key signaling pathway that translates hemolysis into inflammation, VO, and organ pathology in SCD. Our third aim will examine the role of complement activation in SCD hemolysis, inflammation, and VO. The alternative complement pathway is abnormally activated in SCD and is amplified by phosphatidylserine on the outer leaflet of sickle red blood cells and microparticles. We will test the hypothesis that complement activation on the surface of sickle red blood cells and microparticles stimulates inflammation, coagulation, hemolysis, and VO in SCD. Using state-of-the-art molecular techniques including site-directed mutagenesis, bone marrow transplants, cellular/biochemical studies, breeding a unique sickle/Tlr4-deficient mouse model, and in vivo vascular imaging in murine models of SCD, we will provide the foundation for the development of new therapies targeting multiple pathways to interrupt SCD pathophysiology. Understanding TLR4 and complement activation in SCD is expected to lead to therapies targeting the heme-binding site on the MD-2/TLR4 complex and complement inhibitors that will improve the quality of life of SCD patients.